Voted the worlds worst population DNA study.

When this study was first sent to print, it was universally lambasted for being, well, just stupid.

The basic premise of it was that because Greeks share an HLA gene with Ethiopians they were originally of sub Saharan origin. Since this went against every other DNA study that had been done on the Greeks, you think the people involved might have engaged thier brains a bit before coming to that conclusion…

HLA alleles have been determined in individuals from the Re-public of Macedonia by DNA typing and sequencing. HLA-A, -B, -DR, -DQ allele frequencies and extended haplotypes have been for the first time determined and the results compared to those of other Mediterraneans, par-ticularly with their neighbouring Greeks. Genetic distances, neighbor-join-ing dendrograms and correspondence analysis have been performed. The following conclusions have been reached: 1) Macedonians belong to the ‘‘older’’ Mediterranean substratum, like Iberians (including Basques), North Africans, Italians, French, Cretans, Jews, Lebanese, Turks (Anatolians), Ar-menians and Iranians, 2) Macedonians are not related with geographically close Greeks, who do not belong to the ‘‘older’’ Mediterranenan substratum, 3) Greeks are found to have a substantial relatedness to sub-Saharan (Ethiopian) people, which separate them from other Mediterranean groups. Both Greeks and Ethiopians share quasi-specific DRB1 alleles, such as *0305, *0307, *0411, *0413, *0416, *0417, *0420, *1110, *1112, *1304 and *1310. Genetic distances are closer between Greeks and Ethiopian/sub-Saharan groups than to any other Mediterranean group and finally Greeks cluster with Ethiopians/sub-Saharans in both neighbour joining dendrograms and correspondence analyses. The time period when these relationships might have occurred was ancient but uncertain and might be related to the displacement of Egyptian-Ethiopian people living in pharaonic Egypt.

The highly polymorphic HLA system has been validated as useful for distinguishing and/or relating populations (and individuals) in many research studies since the first International HLA Anthropology Workshop (Evian, 1973) and in all the subsequent seven International Workshops. HLA gene frequencies correlate with geographically related populations. The existence or absence of gene flow among neighbouring ethnic groups may be assessed with the study of HLA frequencies and the corresponding genetic distances (1, 2).

Ancient Macedonians were among the peoples that lived between northern Greece (Thessaly) and Thrace in the Balkans and were considered by the classical Greeks as ‘‘non-Greek barbarians’’ that could not participate in the Greek Olympic Games (3). Herodotus wrote that ‘‘Macedonians’’ were ‘‘Dorians’’ and were never admitted to the Greek community (4). They did not speak Greek but another language presently unknown and of which only proper names remain; nowadays, they speak a Slavic language (5). Macedonians fought against the Greeks between 357-336 B.C. under King Philip II. They defeated the Greeks at the Battle of Chaironea (338 B.C.). The Macedonian empire extended from the Balkan Peninsula to the Himalayas and to North Africa during the reign of Philip’s son, Alexander the Great (6). Thereafter, Macedonia was conquered by the Romans and has been disputed in more recent times by Serbs and/or Bulgars. Ottoman Turks controlled Macedonia between 1380-1912 A.D., and it was integrated into Yugoslavia in 1946. In 1991, after the partition of Yugoslavia, a referendum gave Macedonia its independence. The present ethnic groups within the country are: 1) Macedonians: 1,279,000; 2) Albanians: 377,000; 3) Turks: 87,000; 4) Serbs: 44,000; and 5) others: 40,000. The northern-most region of Greece is also known as Macedonia and this is why Greece has opposed the independence of the country while it bears the same name (7).

Furthermore, we have found that the Greeks did not cluster together with other Mediterranean populations, including both western (Iberians, Algerians, Berbers) and eastern (Cretans, Jews, Lebanese, Egyptian, Turks-Anatolians) Mediterraneans (8–10).

The aim of the present work is to determine the relative contributions of Macedonians and Greeks to the present-day genetic pool of Mediterranean peoples. For these purpose, both HLA class I and class II DNA typings have been studied in Macedonians for the first time. The genetic relationship of Macedonians and Greeks to other Mediterraneans, including North Africans (Berbers from Agadir and El Jadida areas and Algerians from Algiers), Iberians (Spaniards, Basques and Portuguese) and Greeks (from Attica, Aegean and Cyprus) were calculated. In addition, sub-Saharan and other Africans were compared with all available Mediterranean groups in order to solve the question of the unique Greek HLA profile.

Material and methods

Population samples

Samples from one hundred and seventy-two unrelated Macedonians in Skopje (Institute of Blood Transfusion, Tissue Typing Laboratory), the Republic of Macedonia capital, were used for HLA geno-typing and phylogenetic calculations. All were Macedonian language speakers and their ancestors did not belong to a country minority group (detailed above). The origin of all other populations used for comparisons is given in

Generic HLA class I (A and B) and high-resolution HLA class II (DRB1 and DQB1) genotyping was performed using a reverse dot-blot technique with the Automated Innolipa system (Innogenetics N.V., Zwijndrecht, Belgium). HLA-A, -B, -DRB1, and -DQB1 allele DNA sequencing was only done when indirect DNA typing (reverse dot-blot) yielded ambiguous results (11). Statistical analysis was performed with Arlequin v1.1 software kindly provided by Ex-coffier and Slatkin (12). In summary, this program calculated HLA-A, -B, -DRB1 and -DQB1 allele frequencies, Hardy-Weinberg equilibrium and the linkage disequilibrium between two alleles at two different loci. Linkage disequilibrium (D¿; also named LD, see ref. 13) and its level of significance (P)for2À2 comparisons were deter-mined using the formulae of Mattiuz and co-workers (14) and the 11th International Histocompatibility Workshop methodology (13). In addition, the most frequent complete haplotypes were deduced following a methodology used in the 11th International Histocompatibility Workshop: 1) the 2, 3, and 4 HLA loci haplotype frequencies (2, 15, 16); 2) the haplotypes previously described in other populations (2, 16); and 3) haplotypes which were assigned if they appeared in two or more individuals and the alternative haplotype was well defined. In order to compare allelic and haplotype HLA frequencies with other populations, the reference tables used were those of the 11th and 12th International HLA Workshops (2, 16; see also Table 1). Phylogenetic trees (dendrograms) were constructed with the allelic frequencies by applying the Neighbor-Joining (NJ) method (17) with the genetic distances between populations (DA, 18) and using DISPAN software containing the programs GNKDST and TREEVIEW (19, 20). A three-dimensional correspondence analysis and its bidimensional representation was carried out using the VISTA v5.02 computer program (21, http:/forrest.psych.unc.edu). Correspondence analysis comprises a geometric technique that may be used for displaying a global view of the relationships among populations according to HLA (or other) allele frequencies. This methodology is based on the allelic frequency variance among populations (similarly to the classical principal components methodology) and on the display of a statistical projection of the differences.

Results

Characteristic HLA allele frequencies of the Macedonian population compared to other Mediterraneans. The expected and observed allele frequencies for HLA-A, -B, -DRB1 and -DQB1 loci do not significantly differ and the population sample is in Hardy-Weinberg equilibrium. Table 2 shows the HLA allele frequencies found in the Macedonian population. Fourteen different HLA-A and twenty-eight different HLA-B alleles were observed in the Macedonian population. Six HLA-A alleles and seven HLA-B alleles had frequencies higher than 5% (A*01, A*02, A*03, A*11, A*24, A*26, B*07, B*08, B*18, B*35, B*38, B*44 and B*51) and these are characteristic of Mediterranean populations (8–10, 22).
______________________________________________________________
Genetic distances between populations (DA) between Macedonians and other populations

With regard to the HLA class II alleles, thirty-one different DRB1 alleles were found and only six had frequencies higher than 5%; DQ allele frequencies reflect the DRB1 locus allele distribution due to the strong linkage disequilibrium between these two loci. Two types of analyses were carried out to compare Macedonian HLA frequencies with other Mediterranean population frequencies: 1) with DRB1 data, which is probably a more informative and discriminating methodology; and 2) with generic (low-resolution) DR-DQ data. These two types of analysis were both performed because some of the populations used for comparison lacked HLA-A and -B data [Berbers (from Souss, Agadir area, Morocco), Jews (Ashkenazi), Jews (Morocco), Jews (non-Ashkenazi), Lebanese (NS and KZ), see Table 1], or high resolution HLA-DQ data [(Greeks (Attica), Greeks (Cyprus), Greeks (Attica-Aegean), see Table 1]], or only generic HLA-DR and -DQ data were available [Portuguese, Turks, Iranians, Armenians and Egyptians, see Table 1]. These partially HLA-typed populations should have been ignored, but they could be analyzed conjointly taking into account only either DRB1 or generic DR and DQ frequencies (Tables 3, 6, Figs 1–3). Analyses using DRB1and DQB1 conjointly were made but are not shown because only a few populations could be used and the results are concordant with the DRB1 analysis. Finally, it should be pointed out that class I generic typing tends to homogenize the comparisons based on DRB1 high-resolution typing (see ref. 22); one class I allele obtained by generic DNA typing may contain several class I alleles, while this is not the case for most DRB1 alleles. Fig. 1 depicts an HLA class II (DRB1) neighbor-joining tree. Populations are grouped into three main branches with high boots-trap values: the first one groups both eastern (including Macedonians, Cretans, Jews, Lebanese) and western Mediterraneans (Europeans and North Africans; Sardinians are also included in the first group). The second branch is formed by African Negroid populations and the third one includes Greek and sub-Saharan populations. This distribution is also confirmed in the correspondence analysis (Fig. 2): the three groups are clearly delimited and a west to east Mediterranean gradient is shown. The Macedonian population shows the closest genetic distance with Cretans (Table 3) and no discontinuity is observed with eastern and western Mediterraneans reflecting the genetic similarity among these populations. It is evidenced that Cretans-Greeks distance is high. These results are con-firmed using DR and DQ generic typings (see Fig. 3 and data not shown) which were used in order to include other Mediterranean populations (Iranians, Armenians, Egyptians and Turks, see Table 1). A DR-DQ neighbour-joining tree (data not shown) maintains the West to East Mediterranean gradient and also the group formed by Greeks and sub-Saharan populations. Turks (old Anatolians), Kurds, Iranians and Armenians have been shown specifically to cluster with the eastern Mediterranean groups (Arnaiz-Villena et al., submitted). On the other hand, genetic distances obtained by using DR-DQ generic typing allele frequencies (data not shown) show that Iranians (1.10À10 »2 ) and Cretans (1.54À10 »2 ) are the two populations closest to the Macedonians followed by the other Mediterranean populations. A discontinuity is found between Berbers (Souss) and Greeks (Attica) (9.59À10 »2 vs. 12.42À10 »2) showing that the latter have a distant relationship with Mediterranean populations as previously described (10, 22) and cluster together with the sub-Saharan populations.

______________________________________________________________
Most frequent HLA-A, -B, -DRB1, and -DQB1 extended haplotypes in the Macedonian

Extended HLA haplotypes were determined in Macedonians and compared with those previously reported in other populations (Table 4). HLA-A-B and DRB1*-DQB1* two-loci linkage disequilibrium data (not shown) show that the most frequent combinations are characteristic of European and Mediterranean (western and eastern) populations (B*18-DRB1*1104, Haplotype Frequency (HF): 9.0; A*02-B*18, HF: 8.1; A*01-B*08, HF: 5.5; B*08-DRB1*0301, HF: 5.2; A*24-B*35, HF: 4.9 and B*07-DRB1*1501, HF: 4.1). The HLA-A-B-DR-DQ extended haplotypes found in the Macedonian population (Table 4) reflect common characteristics with the other ‘‘older’’ Mediterranean background (see footnote to Table 4). These haplotype results are concordant with those obtained by the allele frequency analyses (genetic distances, neighbor-joining trees and correspondence, see above).

Common alleles of Greeks with sub-Saharan Africans

In order to study the possible origin of the Greeks who remain outliers among Mediterraneans (10, 22), specific DRB1 alleles present in Greeks and not present in the other Mediterranean populations were searched in other geographically not very distant populations. Our own data, the 11th and 12th International Histocompatibility Workshops reference panels (2, 16, 23) and other previously described data were used (see Table 1). Table 5 shows the presence of these Greek alleles mainly in sub-Saharan populations from Ethiopia (Amhara, Oromo), Sudan (Nuba) and West Africa (Rimaibe, Fulani, Mossi).

Some of these alleles are sporadically present in other populations without any relationships among them (see footnote to Table 5). It may be deduced from these data that sub-Saharans and Greeks share quasi-specific HLA-DRB1 alleles. The neighbor-joining tree (Fig. 1) and the correspondence analyses (Figs 2 and 3) confirm this Greek/sub-Saharan relatedness. The HLA-DRB1 genetic distances between Greeks and other Mediterraneans are shown in Table 6 and also support a sub-Saharan/Greek relatedness; genetic distances with HLA-DR and -DQ generic typings (not shown) give essentially the same results. No relationship of Greeks is seen with the Senegalese and South African Blacks (Bantu and people coming from the Guinea Gulf after the Bantu expansion, respectively (24)), nor with the present day Bushmen (24). Two different types of problem regarding the obtained data are discarded: 1) mistakes in the HLA typings and 2) mistakes in the assignation of these specific alleles (DRB1*0417, *1112, etc, see Table 5). These problems are not likely to exist in the present work because; 1) HLA typings have been made by genetic technologies in three different Greek populations (2, 23) and 2) similar results are obtained when generic typing is used (DR-DQ analysis in

Macedonians

Our results show that Macedonians are related to other Mediterraneans and do not show a close relationship with Greeks; however they do with Cretans (Tables 3, 4, Figs 1–3). This supports the theory that Macedonians are one of the most ancient peoples existing in the Balkan peninsula, probably long before arrival of the Mycaenian Greeks (10) about 2000 B.C. Other possible explanation is that they might have shared a genetic background with the Greeks before an hypothetical admixture between Greeks and sub-Saharas might have occurred. The cultural, historical and genetic identity of Macedonians is established according to our results. However, 19th century historians focused all the culture in Greece ignoring all the other Mediterranean cultures present in the area long before the classical Greek one (25). Greeks are genetically related to sub-Saharans

Much to our surprise, the reason why Greeks did not show a close relatedness with all the other Mediterraneans analyzed (Tables 5, 6 and Figs 1–3) was their genetic relationship with sub-Saharan ethnic groups now residing in Ethiopia, Sudan and West Africa (Burki-na-Fasso). Although some Greek DRB1 alleles are not completely specific of the Greek/sub-Saharan sharing, the list of alleles (Table 5) is self-explanatory. The conclusion is that part of the Greek genetic pool may be sub-Saharan and that the admixture has occurred at an uncertain but ancient time. The origin of the West African Black ethnic groups (Fulani, Mossi and Rimaibe sampled in Burkina-Fasso) is probably Ethiopian (26, 27) (Fig. 4). The Fulani are semi-nomadic hunters and gatherers and one of the few people in the area to use cows’ milk and its by-products to feed themselves and to trade; their facial parameters show a Caucasian admixture. The Rimaibe Blacks have been slaves belonging to the Fulani and have frequently mixed with them (27). The Nuba people are now widespread all over Sudan, but are descendants of the ancient Nubians that ruled Egypt between 8th–7th centuries B.C. (28) and later established their kingdom at Meroe, North Khartoum. Two kinds of Nubians were described in ancient times: Reds and Blacks, probably reflecting the degree of Caucasian admixture. Both the Oromo and Amharic peoples live in the Ethiopian mountains (27). They obviously have in common a genetic back-ground with the west-African groups mentioned above. Linguistic, social, traditional and historical evidence supports an east-to-west migration of peoples through the Sahel (southern Sahara strip), although this is still debated (26, 27). Thus, it is hypothesized that there could have been a migration from southern Sahara which mixed with ancient Greeks to give rise to a part of the present day Greek genetic background.

The admixture must have occurred in the Aegean Islands and Athens area at least (Figs 1 and 2). The reason why this admixture is not seen in Crete is unclear but may be related to the influential and strong Minoan empire which hindered foreigners establishment (10).

Also, the time when admixture occurred could be after the overthrown of some of the Negroid Egyptian dynasties (Nubian or from other periods) or after undetermined natural catastrophes (i.e.: dryness). Indeed, ancient Greeks believed that their religion and culture came from Egypt (4, 25).

This paper was swifly criticized by this letter from some of the biggest names in population genetics..

Sir – Even though the controversial withdrawal of a paper on the genetic relatedness of Palestinians and Jews by the journal Human Immunology (see Nature 414, 382; 2001) is a minor episode compared with the tragedies caused by ethnic/religious conflicts over past decades, the issues involved are worth revisiting.

The stated purpose of the paper by Antonio Arnaiz-Villena et al. was to “examine the genetic relationships between the Palestinians and their neighbours (particularly the Jews) in order to: (1) discover the Palestinian origins, and (2) explain the historic basis of the present … conflict between Palestinians and other Muslim countries with Israelite Jews”.
They conclude: “Jews and Palestinians share a very similar HLA genetic pool that supports a common ancient Canaanite origin. Therefore, the origin of the long-lasting Jewish–Palestinian hostility is the fight for land in ancient times.”

It is difficult to believe that knowledge of genes may help to explain the present conflict.

Although population genetics can address issues of relatedness of populations, mating patterns, migrations and so on, obviously it cannot provide evidence about reasons for conflicts between people.

Our primary concern, however, is that the authors might be perceived to have been discriminated against for political, as opposed to legitimate scientific, reasons. Even a cursory look at the paper’s diagrams and trees immediately indicates that the authors make some extraordinary claims. They used a single genetic marker, HLA DRB1, for their analysis to construct a genealogical tree and map of 28 populations from Europe, the Middle East, Africa and Japan. Using results from the analysis of a single marker, particularly one likely to have undergone selection, for the purpose of reconstructing genealogies is unreliable and unacceptable practice in population genetics.

The limitations are made evident by the authors’ extraordinary observations that Greeks are very similar to Ethiopians and east Africans but very distant from other south Europeans; and that the Japanese are nearly identical to west and south Africans. :) It is surprising that the authors were not puzzled by these anomalous results, which contradict history, geography, anthropology and all prior population-genetic studies of these groups.

Surely the ordinary process of refereeing would have saved the field from this dispute. We believe that the paper should have been refused for publication on the simple grounds that it lacked scientific merit.

Neil Risch
Department of Genetics, Stanford University School of Medicine, Stanford, California

L. Luca Cavalli-Sforza
Department of Genetics, Stanford University School of Medicine, Stanford, California

By now, I’m reasonably familiar with the DNA studies they are referring to. Sub Saharan African mitochondrial DNA and Y chromosomes are only found at trace in Greece, and in every other study Greeks cluster with Europeans. This one gene (part of the immune system) could very easily be explained by one Ethiopian making it to Greece in classical times and natural selection taking over. It could be indicative of better gluten tolerance, a resistance to measles, or just about anything. Also, the neutrality of the scientist heading the project was in dispute.

In the present study we analyzed for the first time HLA class I and class II polymorphisms defined by high-resolution typing methods in the Bulgarian population. Comparisons with other populations of common historical background were performed. Most HLA-A, -B, -DRB alleles and haplotypes observed in the Bulgarian population are also common in Europe. Alleles and haplotypes considered as Mediterranean are relatively frequent in the Bulgarian population. Observation of Oriental alleles confirms the contribution of Asians to the genetic diversity of Bulgarians. The use of high-resolution typing methods allowed to identify allele variants rare for Europeans that were correlated to specific population groups. Phylogenetic and correspondence analyses showed that Bulgarians are more closely related to Macedonians, Greeks, and Romanians than to other European populations and Middle Eastern people living near the Mediterranean. The HLA-A,-B,-DRB1 allele and haplotype diversity defined by high-resolution DNA methods confirm that the Bulgarian population is characterized by features of southern European anthropological type with some influence of additional ethnic groups. Implementation of high-resolution typing methods allows a significantly wider spectrum of HLA variation to be detected, including rare alleles and haplotypes, and further clarifies the origin of Bulgarians.

Institute of Immunobiology and Human Genetics, Faculty of Medicine, University Ss. Kiril and Metodij, Skopje, Macedonia.

The Macedonian population is of special interest for HLA anthropological study in the light of unanswered questions regarding its origin and relationship with other populations, especially the neighbouring Balkanians. Two studies have been performed to examine HLA molecular polymorphism in the Macedonian population, so far. The present study is the first to be performed in Macedonia using high-resolution sequence-based method for direct HLA typing. The study included 158 unrelated healthy volunteers of Macedonian origin and nationality, having a Christian Orthodox religion. After the simultaneous amplification of exon-2 on both HLA-DRB1 alleles, DNA sequencing was used for genotype assignment. In the 158 samples analysed, all 316 alleles were typed and a total of 29 different DRB1 alleles were detected, with DRB1*1601 being the most frequent allele (14.9%), followed by DRB1*1104 (13.9%). A phylogenetic tree constructed on the basis of the high-resolution data deriving from other populations revealed the clustering of Macedonians together with other Balkan populations (Greeks, Croats, Turks and Romanians) and Sardinians, close to another “European” cluster consisting of the Italian, French, Danish, Polish and Spanish populations. The included African populations grouped on the opposite side of the tree

Allelic frequencies of 182 tri- and tetra-autosomal microsatellites were used to examine phylogenetic relationships among 19 extant human populations. In particular, because the languages of the Basques and Hunza Burusho have been suggested to have an ancient relationship, this study sought to explore the genetic relationship between these two major language isolate populations and to compare them with other human populations. The work presented here shows that the microsatellite allelic diversity and the number of unique alleles were highest in sub-Saharan Africans. Neighbor-joining trees based on genetic distances and principal component analyses separated populations from different continents, and are consistent with an African origin for modern humans. For the first time, with biparentally transmitted markers, the microsatellite tree also shows that the San are the first branch of the human tree before the branch leading to all other Africans. In contrast to an earlier study, these results provided no evidence of a genetic relationship among the two language isolate groups. Genetic relationships, as ascertained by these microsatellites, are dictated primarily by geographic proximity rather than by remote linguistic origin, Mantel test, R(0) = 0.484, g = 3.802 (critical g value = 1.645; P = 0.05). Copyright 2003 Wiley-Liss, Inc.

As you can see, the Greeks are very similar to other European populations.